Thesis (M.S.)--University of Michigan, 1967.

Thesis (M.S.)--University of Michigan, 1967.

Thesis--University of Helsinki, 1978.

Thesis--University of Helsinki, 1978.

Connective tissue initially arises from embryonic mesenchymal stem cells (MSC) that originate from the mesoderm during embryogenesis and are capable of differentiating into connective tissue lineages such as adipocytes, osteoblasts, chondrocytes and fibroblasts. Connective tissue is composed of cells held together by the extracellular matrix (ECM). The fibrillins and latent transforming growth factor binding proteins form a superfamily of ECM proteins characterised by the presence of a unique domain, the 8- cysteine transforming growth factor beta binding domain (TGFß). The fibrillin proteins (fibrillin-1, fibrillin-2 and fibrillin-3 in most vertebrates, encoded by the FBN1, FBN2 and FBN3 genes respectively), are major components of the 10nM microfibrils found in ECM of many tissue types, for example mesenchyme-derived connective tissues. Fibrillin-1 and fibrillin-2 are also thought to be required for stabilization and storage of latent TGFβ complexes. Mutations in FBN1 cause Marfan syndrome, a connective tissue disorder characterised by abnormalities in the microfibrils resulting in musculoskeletal, ocular, cardiovascular and other complications. FBN2 mutations lead to congenital contractural arachnodactyly, which has a musculoskeletal phenotype similar to Marfan syndrome. There are currently no known diseases associated with FBN3 mutations. In this project, the expression of fibrillins was investigated using human cell lines during early development, mesenchymal stem cell differentiation and in further differentiated mesenchymal cell lines, for example in osteocytes (osteosarcomas), chondrocytes and fibroblast lineage. Immunocytochemistry was used to examine protein expression, real-time PCR and expression microarrays to determine mRNA synthesis and RNAi suppression of gene expression to determine possible functions of fibrillins and associated ECM proteins. In addition, a genome wide bioinformatics evaluation was performed of transcription start sites for the fibrillin gene family utilising the information obtained from the FANTOM5 consortium. The three fibrillin genes showed differing expression patterns in cell lines depending on the stage of development/differentiation. During embryogenesis, expression of FBN3, FBN2 and FBN1 increased sequentially in that order. Expression of FBN3 followed the same pattern as expression of known pluripotency markers, while expression of FBN2 correlated with expression of markers for later stages of mesoderm differentiation. FBN1 expression was associated with mesenchymal markers, and this was supported by a study of mesenchymal stem cells differentiation to the adipose lineage. Fibrillin-1 microfibrils and RNA expression were present early in primary adult human MSC differentiating to adipocytes, suggesting that a fibrillin matrix is required for initial MSC attachment. As differentiation proceeded, fibrillin -1 expression decreased, with rapid degradation of the microfibrils. Fibrillin-2 expression increased following differentiation and fibrillin-3 was not expressed. These results suggest that fibrillin-1 plays an important structural and regulatory role in the early stages of connective tissue development but is not required to maintain the differentiated state. Many genes showed the same expression pattern as FBN1. To better understand the importance of fibrillin-1 and its interaction with these coexpressing genes, fibrillin-1 was knocked down using siRNA in fibroblast, chondrocyte and osteosarcoma cell lines. There were little to no effects identified in chondrocyte and osteosarcoma cell lines, and only a few genes were altered following the reduction of fibrillin-1 mRNA in fibroblasts, suggesting that fibrillin-1 is not a central regulator but an endpoint. This was surprising given its potential role in controlling bioavailability of TGFβ, a key regulator of mesenchymal cells. In addition, the evolution of the fibrillin gene family was studied and it was found that the gene structure, amino acid sequence and genomic positions of each gene are widely conserved across vertebrates, suggesting an important role in vertebrate body structure. However, the differences in gene structure and sequence between the three fibrillin genes suggest divergent function. Fibrillin-1 mutations with the most severe phenotypes are located in regions that are highly conserved. This study shows that there is a clear developmental and evolutionary distinction between the three fibrillins. Fibrillin-3 was associated with pluripotency and its presence in differentiating foetal liver and brain may suggest that there are residual pluripotent cells in these developing tissues. Fibrillin-2 appeared to be a marker for the mesodermal stage and its role in adult cells is currently not clear. Fibrillin-1 was present in cells already predetermined to go to mesenchymal lineages, but it was minimal in the advanced stages of differentiation suggesting that it may be a marker for relatively plastic mesenchymal cells prior to commitment to a specific lineage. These results will assist in the understanding of disorders resulting from fibrillin gene mutations and have identified coexpressed proteins, potential modifiers that could be the targets of gene therapy and candidates for similar connective tissue.

No description available.

published_or_final_version / Biochemistry / Master / Master of Philosophy

Cystein rich protein 61 (CYR61/CCN1) und Connective tissue growth factor (CTGF/CCN2) stellen aufgrund ihrer Multifunktionalität zwei sehr interessante Vertreter aus der derzeit sechs Mitglieder umfassenden Familie der CCN-Proteine (CCN- CYR61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1-3/CCN4-6) dar. Seit der Entdeckung von CYR61 und CTGF konnten die überlappenden, aber meist nicht redundanten zellspezifischen Effekte in verschiedenen Zellsystemen nachgewiesen werden. Die Einflüsse auf zahlreiche Prozesse wie Proliferation und Migration, aber auch Angiogenese und das Überleben von Zellen lassen eine weitreichende Bedeutung im Zusammenhang mit vielen Entwicklungsprozessen vermuten, so auch der des muskuloskelettalen Systems und der Entwicklung der Lunge. In der vorliegenden Arbeit wurden für die nähere Charakterisierung von CYR61 und CTGF humane mesenchymale Stammzellen (hMSC) und die humane primäre Lungenendothelzelllinie HPMEC-ST1.6R (human pulmonary microvascular endothelial cells) gewählt. Beide Zellsysteme sind für die Untersuchung der Funktionsfähigkeit in den verschiedenen Kompartimenten bestens geeignet. So ist die Zelllinie HPMEC-ST1.6R den primären Endothelzellen, im Vergleich mit anderen in der Forschung eingesetzten Zelllinien, in Bezug auf spezifische Oberflächenmarker am nächsten. Mesenchymale Stammzellen bilden als multipotente Zellen das Rückrat des muskuloskelettalen Systems und sind an der Homöostase des menschlichen Stütz- und Bewegungsapparates maßgeblich beteiligt. Um experimentell nutzbare Konzentrationen an rekombinanten Proteinen zu erhalten, wurde ein Baculovirus-Expressionsystems gewählt. Nach der erfolgreichen Klonierung der CTGF/Fc-Tag Sequenz in einen Expressionsvektor konnte dies auch durch Produktion in SF21-Insektenzellen erreicht und erstmalig rekombinantes CTGF/Fc von hoher Reinheit gewonnen werden. Allerdings konnte eine beständige Funktionsfähigkeit der aufgereinigten Proteine mittels eines Proliferationstestes nachfolgend nur bedingt bestätigt werden. Für die weitere Versuchsplanung, einer Untersuchung der Auswirkung von rekombinantem CTGF (rCTGF) bzw. CYR61 (rCYR61) auf die Zielzellen, musste zunächst die zelleigene ctgf bzw. cyr61 Expression herunterreguliert werden, um einen endogenen Störeffekt auszuschließen. Durch den Einsatz spezifischer shRNAs konnte ctgf/CTGF sowohl in den hMSC-, wie auch den HPMEC-ST1.6R-Zielzellen deutlich herunterreguliert und nachfolgend eine markant reduzierte Proliferation beobachtet werden. Ein Effekt für die Regulation von cyr61 blieb aus. In dieser Arbeit wurden anschließend erstmals mittels Microarray-Analysen Veränderungen im Genexpressionsmuster der ctgf herunterregulierten hMSC- bzw. Lungenendothelzellen gegenüber Kontrollzellen untersucht. Des Weiteren war die Auswirkung einer Behandlung von ctgf herunterregulierten Zielzellen mit rCTGF gegenüber unbehandelten Kontrollzellen von Interesse. Für beide Zellsysteme konnten signifikante Genregulationen nach der Behandlung mit CTGF spezifischen shRNAs gegenüber den Kontrollzellen detektiert werden, mit interessanten Genclustern im Bereich der TGF-beta (transforming growth factor ß) Signalgebung, sowie der fokalen Adhäsion (z.B. VEGF). Eine Behandlung mit rCTGF hingegen zeigte gegenüber den unbehandelten Kontrollzellen in der Auswertung der Microarray-Analyse keine signifikante Veränderung im Genexpressionsmuster. In dieser Arbeit wurden, neben einer effektiven Gewinnung von rekombinantem CTGF und der Herunterregulation der endogenen ctgf Expression, wichtige Erkenntnisse zur Biologie von CTGF (und CYR61) in mesenchymalen Stammzellen hMSC und der Lungenendothelzelllinie HPMEC-ST1.6R erlangt. Die erhaltenen Microarray-Daten bieten eine fundierte Grundlage für zahlreiche fortführende Untersuchungen. / Cystein rich protein 61 (CYR61/CCN1) and connective tissue growth factor (CTGF/CCN2) are two very interesting members of the CNN family (CCN- CYR61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1-3/CCN4-6) consisting of six members so far. Since its discovery the overlapping, but mostly non-redundant effects of CYR61 and CTGF were shown in different cell systems. Both proteins are linked to many different processes like proliferation and migration, but also angiogenesis and survival. They seem to be involved in very fundamental biological processes, amongst other the development of the musculoskeletal system and the lung and were analyzed in this study. To distinguish the two proteins CYR61 and CTGF, primary human mesenchymal stem cells (hMSC) and a human pulmonary endothelial cell line (HPMEC-ST1.6R) were chosen. Both cell systems are suited very well for getting more information about the function in these different compartments. So the cell line HPMEC-ST1.6R is more related to primary endothelial cells in reference to the cell surface markers, compared to other cell lines used for experimental research. Mesenchymal stem cells form the backbone of the musculoskeletal system and are involved in the homeostasis of this complex system. Getting adequate concentrations of recombinant proteins for the upcoming experiments a baculovirus expression system was chosen. After successful cloning of the CTGF/Fc-Tag sequence into an expression vector, recombinant CTGF/Fc of high purity was obtained for the first time, produced in SF21 insect cells. However the stable functioning of the proteins was partly confirmed by proliferation tests. To study the effect of recombinant CTGF or CYR61 in further experiments, the endogenous ctgf or cyr61expression had to be downregulated to avoid negative effects. By using specific shRNAs ctgf/CTGF has been downregulated in hMSC as well as HPMEC-ST1.6R cells and subsequently a reduced proliferation was observed. No effect was detected for the regulation of cyr61. In this study for the first time changes in regulation of gene expression after downregulation of ctgf in hMSC and HPMEC-ST1.6R cells were studied by microarray analyses. Furthermore to discover the effect of treating ctgf downregulated cells with recombinant CTGF compared to control cells was another aim of this experimental series. For both cell systems, significant gene regulations were detected after treatment with CTGF specific shRNAs with interesting gene cluster for TGFß-signaling as well as focal adhesion (e.g. VEGF). In contrast, no significant change in gene regulation was detected by microarray analysis after treating the target cells with rCTGF compared to non-treated cells. In summary, besides the effective preparation of rCTGF and the marked downregulation of ctgf gene expression, this study provides fundamental information about CTGF and its biology in hMSC and HPMEC-ST1.6R cells, as well. Based on the numerous detected gene regulations in the microarray analyses the study provides a basis for further experiments.

This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.

This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.